The blood-brain barrier (BBB) is the principal interface between blood and the interstitial fluid that bathes neurons within the brain parenchyma (Abbott et al., Neurobiol Dis. 2010 January; 37(1):13-25). The BBB is formed by highly specialized endothelial cells that maintain an optimal environment for neuronal function by eliminating toxic substances and supplying the brain with nutrients and other metabolic requirements. The BBB likewise presents a formidable obstacle for the systemic delivery of many potentially important therapeutic and diagnostics agents. With the exception of small, lipophilic molecules (MW less than 500 Daltons), which can cross the BBB by transmembrane diffusion, nearly all hydrophilic small molecules, peptides, proteins, RNAs and genetic vectors that could be of therapeutic value are excluded (Pardridge, J Cereb Blood Flow Metab. 2012 November; 32(11):1959-72.). Many of the antibodies designed to treat a variety of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease and frontotemporal dementia will be limited by their inability to reach the pathological target within the brain. Thus, despite tremendous progress in the discovery of potential therapeutics for CNS diseases, very few will be successfully developed without an effective means of delivery across the BBB.
Although the BBB restricts the passage of many substances, brain capillaries use membrane transport systems to deliver important nutrients and macromolecules important for normal brain function. The main route whereby large molecules, such as proteins and peptides, enter the CNS is by the receptor-mediated transcytosis (RMT) which might also be used to shuttle a wide range of therapeutics into the brain in a non-invasive manner (Jones and Shusta, Pharm Res. 2007 September; 24(9):1759-71). Circulating ligands such as transferrin, insulin and leptin interact with specific receptors concentrated on the luminal side of the brain capillary endothelial cells. Once bound to the receptor, the process of endocytosis is initiated as the receptor-ligand complexes cluster and intracellular transport vesicles detach from the membrane (Tuma and Hubbard, Physiol Rev. 2003 July; 83(3):871-932). The transport vesicles containing receptor-ligand complexes or dissociated ligands are directed away from the lysosomal compartment and trancytosed to the brain interstitial side of the endothelial cell, where they are released without disrupting the BBB.
One way to exploit endogenous RMT systems for drug delivery is to couple the drug therapeutic of interest to a vector such as an antibody or ligand that targets a particular RMT system. The drug cargo gains access to the brain parenchyma by “piggybacking” on the delivery vector (i.e., a type of “molecular vehicle” and also described as a molecular Trojan horse), which carries it across the BBB (Pardridge, Nat Rev Drug Discov. 2002 February; 1(2):131-9). The transferrin receptor 1 (TfR-1) endocytotic pathway for iron homeostasis has been one of the most extensively characterized systems for drug delivery across the BBB. TfR-1 mediates influx of iron-loaded transferrin from blood to brain in addition to the transcytosis of iron-depleted transferrin in the reverse direction. Transferrin itself has been used as a vehicle for brain delivery, but transferrin conjugates have to compete for the receptor with the high plasma concentration of the endogenous ligand. The OX-26 mouse monoclonal antibody, which specifically binds the rat transferrin receptor in brain capillaries without blocking the binding of transferrin (Jefferies et al., 1985), was the first antibody used to carry a drug cross the BBB (Freiden et al., Proc Natl Acad Sci USA. 1991 Jun. 1; 88(11):4771-5).
Anti-TfR antibodies have since been modified in a several different ways to deliver heterologous biomolecules, e.g., drug cargo, to the brain. Potential biotechnology products, including lysosomal enzymes, neurotrophins, decoy receptors, antibody fragments have been fused to the carboxyl terminus of the Fc domain of TfR for CNS delivery (Pardrige and Boado, Methods Enzymol. 2012; 503:269-92). More recently, bispecific antibodies have been produced by knobs-into-holes technology whereby one half of the antibody binds the CNS target and the other binds the TfR-1 (Yu et al., Sci Transl Med. 2011 May 25; 3(84):84ra44). Bispecific antibodies have also been generated by fusing the ScFv portion of a TfR-1 antibody to the carboxyl terminus of a therapeutic antibody (Niewoehner et al., Neuron. 2014 Jan. 8; 81(1):49-60) which maintains avid binding to the target. Each of these approaches has provided evidence of CNS activity in animal models following the intravenous injection, indicating that TfR-1 antibodies as therapeutic carriers hold significant promise for the non-invasive treatment of CNS disorders.
Despite these advances, several features of monoclonal antibodies as BBB carriers have hampered their translation from animal to humans. Antibodies are large molecules composed of 4 disulfide-linked subunits that are challenging to format as bispecific molecules. Moreover, functional components outside the antigen recognition domain can lead to off-mechanism toxicity, and complement-mediated lysis of TfR-rich reticulocytes has been reported (Couch et al., Sci Transl Med. 2013 May 1; 5(183):183ra57, 1-12). Another drawback is that TfR antibodies used to date are species-specific, which is problematic for preclinical safety testing of potential therapeutic molecules. Surrogate antibodies to the TfR-1 with the same biochemical properties (binding epitope, affinity, avidity and pH sensitivity) and transcytosis activity will be difficult to identify and antibodies that block ligand binding (Crépin et al., Cancer Res. 2010 Jul. 1; 70(13):5497-506) or inhibit transcytosis and deplete surface receptors (Bien-Ly et al., J Exp Med. 2014 Feb. 10; 211(2):233-44) would be unsuitable as BBB carriers due to potential iron deprivation.
To address the drawbacks inherent in full size antibodies as BBB carriers, a panel of species cross-reactive VNARs to TfR-1 have been identified by phage display and selected for brain uptake. VNARs are isolated variable domains derived from the naturally-occurring single chain antibodies found in the shark (Stanfiled et al., Science. 2004 Sep. 17; 305(5691):1770-3.). Their small size (˜12 kDa), high solubility, thermal stability and refolding capacity (Wesolowski et al., Med Microbiol Immunol. 2009 August; 198(3):157-74) simplifies coupling to a monoclonal antibody or other pharmaceutical. Their modularity offers a wide range of therapeutic design and their species cross-reactivity can facilitate the development and clinical translation of brain penetrant therapeutics to treat a broad spectrum of CNS disorders.
Similar problems are encountered in transporting molecules, such as drug substances, across intestinal epithelium of the gut, where transcellular and paracellular routes of transport exist for water and ions but where larger molecules are transported exclusively by transporter molecules in epithelial cell plasma membranes.
Hence, it is desirable to have new molecular tools for efficient and selective delivery of compounds such as biomolecules (e.g., therapeutics and diagnostics) across the BBB to avoid some or all of the problems discussed above. It would thus also be desirable to have new molecular tools for efficient and selective delivery of compounds biomolecules across the cells of the gastrointestinal (GI) tract thereby increasing the oral bioavailability of certain molecules, e.g., drugs, which do not naturally cross the GI tract when delivered in oral form. Moreover, it would be advantageous to have new selective TfR-specific binding compounds, especially ones having one or more advantageous biological properties with therapeutic and/or diagnostic benefit over current anti-TfR antibodies and other regulators of iron transport systems.